Volume 11, Issue 3, Pages 429-442 (March 2018) The Hardy Rubber Tree Genome Provides Insights into the Evolution of Polyisoprene Biosynthesis  Ta-na Wuyun, Lin Wang, Huimin Liu, Xuewen Wang, Liangsheng Zhang, Jeffrey L. Bennetzen, Tiezhu Li, Lirong Yang, Panfeng Liu, Lanying Du, Lu Wang, Mengzhen Huang, Jun Qing, Lili Zhu, Wenquan Bao, Hongguo Li, Qingxin Du, Jingle Zhu, Hong Yang, Shuguang Yang, Hui Liu, Hui Yue, Jiang Hu, Guoliang Yu, Yu Tian, Fan Liang, Jingjing Hu, Depeng Wang, Ruiwen Gao, Dejun Li, Hongyan Du  Molecular Plant  Volume 11, Issue 3, Pages 429-442 (March 2018) DOI: 10.1016/j.molp.2017.11.014 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Characterization of the E. ulmoides Genome. Circos plot of the top 20 longest scaffolds of superscaffolds. (a) Repeat sequence densities shown as the distribution densities from high (red) to low (blue). (b) Non-coding RNAs (ncRNAs), rRNAs, tRNAs, and other ncRNAs are represented by red, green, and gray lines, respectively. (c) Gene model annotation was compared with the NCBI non-redundant protein database for A. chinensis, A. thaliana, D. carota, P. trichocarpa, S. lycopersicum, V. vinifera, and other organisms. The best Blast matches to these species are indicated in red, orange, green, blue, purple, yellow, and gray lines, respectively. (d–i) The orthologous genes in A. chinensis, A. thaliana, D. carota, P. trichocarpa, S. lycopersicum, and V. vinifera, respectively. (j–m) The FPKM of transcript in roots, staminate flower, stems, and leaves were mapped to the sense and antisense strands, shown in red and blue, respectively. (n) GC content. Molecular Plant 2018 11, 429-442DOI: (10.1016/j.molp.2017.11.014) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Evolution and Synteny of the E. ulmoides Genome. (A) The phylogenetic relationship and split-time estimation is based on all single-copy gene families shared by all species used in this analysis. The black number at each node denotes estimated divergence time. The values above each branch denote gene family gain/loss number at each round of genome duplication after diversification from the common ancestor, and the italicized letters along branches indicate different whole-genome duplication events. Bootstrap values for each node are above 50%. The red nodes indicate the known divergence time of Monocots and Dicots, Asterids and Rosids, and A. thaliana and V. vinifera, respectively. (B) Density distributions of the Ks values for homologous genes. The peak values are shown in the insets for A. chinensis, D. carota, P. trichocarpa, S. lycopersicum, E. ulmoides, and V. vinifera. (C) Schematic representation of syntenic genes among E. ulmoides, S. lycopersicum, V. vinifera, and A. chinensis. Gray lines connect matched gene pairs, with one set highlighted in red. The superscaffolds/chromosomes of E. ulmoides, V. vinifera, A. chinensis, and S. lycopersicum were assigned with purple, orange, green, and blue boxes, respectively. (D) A Venn diagram of shared orthologs among five asterid species (E. ulmoides, A. chinensis, D. carota, S. lycopersicum, and S. tuberosum) based on the gene family cluster analysis. Each number in the diagram represents the number of gene families within a group. Molecular Plant 2018 11, 429-442DOI: (10.1016/j.molp.2017.11.014) Copyright © 2017 The Authors Terms and Conditions

Figure 3 The Eu-Rubber Biosynthesis Pathway and Expression Profiles of Genes Involved in the Pathway. Expression level is presented by log2-transformed fragments mapped per kilobase of transcript length per million total mapped reads (log2-FPKM). Asterisks indicate that the gene expression profile was correlated with the Eu-rubber accumulation at the *p < 0.05 and **p < 0.01 levels. Minus sign indicates that the gene expression profile was negatively correlated with the rubber accumulation. The curves in the center area show the Eu-rubber contents in fruits and leaves between April and September. L, leaf; F, fruit. AACT, acetyl-coenzyme A (CoA) C-acetyltransferase; HMGS, hydroxymethylglutaryl-CoA synthase; HMGR, hydroxymethylglutaryl-CoA reductase; MK, mevalonate kinase; PMK, 5-phosphomevalonate kinase; MDC, mevalonate pyrophosphate decarboxylase; DXS, 1-deoxy-d-xylulose 5-phosphate synthase; DXR, 1-deoxy-d-xylulose 5-phosphate reductoisomerase; MCT, 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase; CMK, 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase; MDS, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; HDS, 4-hydroxy-3-methylbut-2-enyl diphosphate synthase; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; IDI, isopentenyl diphosphate isomerase; GPS, geranyl diphosphate synthase; FPS, farnesyl diphosphate synthase; GGPS, geranylgeranyl diphosphate synthase; REF, rubber elongation factor; SRPP, small rubber particle protein; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; MVA, mevalonate; MVAP, mevalonate-5-phosphate; MVAPP, mevalonate-5-diphosphate; G3P, glyceraldehyde 3-phosphate; DXP, 1-deoxy-d-xylulose 5-phosphate; MEP, 2-C-methyl-d-erythritol 4-phosphate; CDP-ME, 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol; CDP-ME2P, 2-phospho-4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol; MEcPP, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate; HMBPP, 4-hydroxy-3-methylbut-2-enyl diphosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate. Molecular Plant 2018 11, 429-442DOI: (10.1016/j.molp.2017.11.014) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Phylogenetic Analyses, Gene Distribution, and Comparison of Rubber Biosynthesis Pathways. (A) Phylogenetic analysis of FPSs from E. ulmoides (Eu) and H. brasiliensis (Hb). Bootstrap values are shown at the nodes. I and II represent clusters I and II, respectively. (B) Phylogenetic analysis of REF/SRPPs from E. ulmoides and other species. Bootstrap values are shown at the nodes. The REF/SRPP family members in H. brasiliensis and E. ulmoides were expanded in clade I (branches in black) and clade II (branches in red), respectively. Eu, E. ulmoides; Hb, H. brasiliensis; AT, A. thaliana; Os, O. sativa. (C) Distribution of EuREF/SRPPs in the E. ulmoides genome. Arrows indicate the orientation of genes. The assembled genome sequences (scaffold) were mapped to the PacBio subreads (contig) and most of the genes on scaffolds were identical with that on contigs. Dashed lines do not indicate the actual length. (D) Comparison of rubber biosynthesis pathways between E. ulmoides and H. brasiliensis. Identical steps are in gray, whereas differences between steps from FPP to TPI in E. ulmoides and steps from FPP to CPI in H. brasiliensis are highlighted with other colors. In both E. ulmoides and H. brasiliensis, MVA is mainly involved in rubber biosynthesis. FPS*: the FPS family in E. ulmoides has evolved into two subfamilies, I and II, for FPP and long chain TPI synthesis, respectively. TPI, trans-polyisoprene; CPI, cis-polyisoprene. Other abbreviations are identical to those in Figure 3. Molecular Plant 2018 11, 429-442DOI: (10.1016/j.molp.2017.11.014) Copyright © 2017 The Authors Terms and Conditions